Proteins: Evolution, and Analysis Lecture 7

1
Proteins Evolution, and AnalysisLecture
7 9/15/2009
2
Chapter 4
(1)
G A V L I M P F W
3
Chapter 4
(2)
4
(3)
5
The Fischer Convention
Absolute configuration about an asymmetric carbon
related to glyceraldehyde ()
D-Glyceraldehyde (-) L-Glyceraldehyde
6
Cahn - Ingold - Prelog system
Can give absolute configuration nomenclature to
multiple chiral centers. Priority Atoms of
higher atomic number bonded to a chiral center
are ranked above those of lower atomic number
with lowest priority away from you R highest to
lowest clockwise, S highest to lowest
counterclockwise SHgtOHgtNH2gtCOOHgtCHOgtCH2OHgtC6H5gtCH
3gtH
7
(No Transcript)
8
Newman Projection

• A projection formula representing the spatial
  arrangement of bonds on two adjacent atoms in a
  molecular entity.
• The structure appears as viewed along the bond
  between these two atoms, and the bonds from them
  to other groups are drawn as projections in the
  plane of the paper.
• The bonds from the atom nearer to the observer
  are drawn so as to meet at the centre of a circle
  representing that atom.
• Those from the further atom are drawn as if
  projecting from behind the circle.

9
The major advantage of the CIP or RS system is
that the chiralities of compounds with multiple
asymmetric centers can be unambiguously described
10
Structural Hierarchy in proteins
11
Overview of Protein Sequencing
(1) Purify Protein
(2) Determine number of PP End group
analysis (Dansyl chloride rxn)
(3) Fragment PP into smaller peptides Enzymes
(Trypsin, Chymotrypsin, etc.) Chemical (CNBr)
(4) Determine the sequence Edman Degradation with
PITC
(5) Assemble a sequence
(6) Elucidate S-S bonds Amino acid composition
12
Long peptides have to be broken to shorter ones
to be sequenced
13
Q9. You must cleave the following peptide into
smaller fragments. Which of the proteases listed
in the table would be likely to yield the most
fragments? The fewest? NMTQGRCKPVNTFVHEPLVDVQNV
CFKE
14
Cyanogen bromide cleavage of a polypeptide
15
Reconstructing the proteins sequence
Specific chemical cleavage reagents
Cleave the large protein using i.e trypsin,
separate fragments and sequence all of them. (We
do not know the order of the fragments!!) Cleave
with a different reagent i.e. Cyanogen Bromide,
separate the fragments and sequence all of them.
Align the fragments with overlapping sequence to
get the overall sequence.
16
Determining the positions of disulfide bond
17
How to assemble a protein sequence
1. Write a blank line for each amino acid in the
sequence starting with the N-terminus. 2. Follow
logically each clue and fill in the blanks. 3.
Identify overlapping fragments and place in
sequence blanks accordingly. 4. Make sure
logically all your amino acids fit into the
logical design of the experiment. 5. Double check
your work.
18
1 2 3 4 5
6 7 8 9 10 11
12 13 14 15 H3N-_-_-_-_-_-_-_-_-_-_-_-
_-_-_-_-COO-

A - T
F
- M - A - T
A- K - F - M

Q - M - A - K
D - I - K - Q - M
G - M - D - I - K Y - R - G -
M Y - R
Cyanogen Bromide (CNBr) Cleaves after Met i.e
M - X D - I - K - Q - M A - T A - K - F -
M Y - R - G - M
Trypsin cleaves after K or R (positively charged
amino acids) Q - M - A - K G - M - D - I - K F -
M - A - T Y - R
19

• Q11. Separate cleavage reactions of a polypeptide
  by CNBr and chymotrypsin yield fragments with the
  following amino acid sequences. What is the the
  sequence of the intact polypeptide?
• CNBr treatment Chymotrypsin
• 1. Arg-Ala-Tyr-Gly-Asn 1. Met-Arg-Ala-Tyr
• 2. Leu-Phe-Met 2. Asp-Met-Leu-Phe
• 3. Asp-Met 3. Gly-Asn

• Q13. Treatment of a polypeptide with
  2-mercaptoethanol yields two PP
• 1. Ala-Val-Cys-Arg-Thr-Gly-Cys-Lys-Asn-Phe-Leu
• 2. Tyr-Lys-Cys-Phe-Arg-His-Thr-Lys-Cys-Ser
• Treatment of the intact PP with trypsin yields
  fragments with the following aa compositions
• 3. (Ala, Arg, Cys2, Ser, Val) 4. (Arg, Cys2,
  Gly, Lys, Thr, Phe)
• 5. (Asn, Leu, Phe) 6. (His, Lys, Thr)
• 7. (Lys, Tyr)

20
Sequencing by Mass Spectrometry
Electrospray Ionization Mass Spectrometry
ESI-MS spectrum of horse heart apomyoglobin
Q. Two successive peaks in the mass spectrum have
measure m/z ratios of 1414.0 and 1542.3. What is
the original apomyoglobin molecule? p1
(Mz)/z p2 (Mz-1)/z-1 M 16,975D (16,951 D in
table 5-1)
21
Tandem Mass Spectrometry in amino acid sequencing
22
(No Transcript)
23
Protein Evolution
Species variation in homologous proteins
The primary structures of a given protein from
related species closely resemble one another. If
one assumes, according to evolutionary theory,
that related species have evolved from a common
ancestor, it follows that each of their proteins
must have likewise evolved from the corresponding
ancestor.
A protein that is well adapted to its function,
that is, one that is not subject to significant
physiological improvement, nevertheless continues
to evolve.
Neutral drift changes not effecting function
24
Homologous proteins (evolutionarily related
proteins)
Compare protein sequences Conserved residues,
i.e invariant residues reflect chemical
necessities. Conserved substitutions,
substitutions with similar chemical properties
(Asp for Glu), (Lys for Arg), (Ile for
Val) Variable regions, no requirement for
chemical reactions etc.
25
Amino acid difference matrix for 26 species of
cytochrome c
26
(No Transcript)
27
(No Transcript)
28
Phylogenetic tree

• Indicates the ancestral relationships among the
  organisms that produced the protein.
• Each branch point indicates a common ancestor.
• Relative evolutionary distances between
  neighboring branch points are expressed as the
  number of amino acid differences per 100 residues
  of the protein.
• PAM units
• or
• Percentage of Accepted Mutations

29
PAM values differ for different
proteins. Although DNA mutates at a assumed
constant rate. Some proteins cannot accept
mutations because the mutations kill the function
of the protein and thus are not viable.
30
Mutation rates appear constant in time
Although insects have shorter generation times
that mammals and many more numbers of
replication, number of mutations appear to be
independent of the number of generations but
dependent upon time
Cytochrome c amino acid differences between
mammals, insects and plants note the similar
distances
31
Evolution through gene duplication

• Many proteins within an organism have sequence
  similarities with other proteins.
• These are called gene or protein families.
• The relatedness among members of a family can
  vary greatly.
• These families arise by gene duplication.
• Once duplicated, individual genes can mutate into
  separate genes.
• Duplicated genes may vary in their chemical
  properties due to mutations.
• These duplicate genes evolve with different
  properties.
• Example the globin family.

32
Genealogy of the globin family

• Hemoglobin
• is an oxygen transport protein
• it must bind and release oxygen as the cells
  require oxygen
• Myoglobin
• is an oxygen storage protein
• it binds oxygen tightly and releases it when
  oxygen concentrations are very low

a2b2 a2g2 a2d2 z2e2
33
The globin family history

• 1. Primordial globin gene acted as an
  Oxygen-storage protein.
• 2. Duplication occurred 1.1 billion years ago.
• lower oxygen-binding affinity, monomeric
  protein.
• 3. Developed a tetrameric structure two a and two
  b chains increased oxygen transport
  capabilities. (a2b2).
• Mammals have fetal hemoglobin with a variant b
• chain i.e. g (a2g2).
• 5. Human embryos contain another hemoglobin
  (z2e2).
• 6. Primates also have a d chain with no known
  unique function. (a2d2).

34
Modular Construction of some proteins
Modules (sequence motifs) 40 -100 residues
35
Lecture 8 (9/17/2009) Chapter 6 - Proteins
3-D structure 6-1. Secondary Structure